PET (Positron Emission Tomography) is a powerful non-invasive molecular imaging technology which provides sufficient sensitivity to visualize and quantitate in vivo physiological interactions in a volume- and target-depth independent manner. PET has been widely used clinically to detect various diseases, such as cancer, cardiovascular disease, and neurodegenerative disease, non-invasively. With a specific positron-emitter labeled molecular probe, PET can reveal the status of specific physiological and biochemical function of living organisms. The system detects pairs of gamma rays emitted at approximately 180 degrees to each other in the event of positron annihilation. With information obtained, three-dimensional images of tracer location and concentration can be reconstructed mathematically to visualize the physiological and functional changes of biological system of interest within the body.
PET tracers are chemical and/or biological molecules labeled with short-lived positron emitting isotopes. The PET tracer is injected into the animal or human body just before scanning. The tracer travels through the body to a target site and its emitted position reveals information about the target receptor density, target protein distribution or specific biological functions, such as metabolism of the radiolabeled molecule. Various PET tracers have been developed to interact with different biological targets and entities according to their biological mechanism in vivo. Some of them have become clinically available and have proven to be useful for disease diagnosis and monitoring therapeutic efficacy, such as 18F-labeled PET tracers (e.g. [18F]FDG, [18F]FLT, and [18F]FDOPA) and 11C-labeled PET tracers (e.g. [11C]acetate, [11C]choline, and [11C]methioine).
Among the available PET radionuclides, fluorine-18 (F-18) is the most widely used in clinical settings. The half-life of C-11 is very short (only 20.4 min) while F-18 has a longer half-life (110 min). F-18 labeled compounds are more desirable because this longer half life allows multi-step radiochemical manipulations as well as in vivo biological studies that can last for several hours. These unique features make fluorine-18 a very attractive isotope for PET imaging because of its ease for mass production using a biomedical cyclotron, well-established synthesis procedures to incorporate F-18 into desired structures, and high-spatial resolutions of resulted images due to its low positron energy. In addition, its high positron abundance and nearly monochromatic emission lead to simplified detection, data processing and greater sensitivity. F-18 is also preferred for the development of novel PET tracers because it is available in high specific activity. The flexibility of fluorine-18 chemistry not only produces large amounts of useful PET tracers originated from small organic molecules but also has potential to turn certain highly-specific targeting biological molecules, such as proteins or peptides (Annexin V, VIP, RGD, anti-CEA diabody, etc.) into valuable PET tracers.
Biomolecules, such as peptides, proteins, antibodies, diabodies, minibodies and others have gained importance as a role in PET tracers. They serve not only as potential therapeutics but also PET imaging probes once labeled with positron-emitters, e.g. F-18. The concept of applying radiolabeled biomolecules to target receptor-(over)expressed tissues in vivo has opened up a new avenue for immunoPET as a very useful diagnostic tool to visualize tumor lesions. However, because of the harsh chemical conditions associated with direct radiofluorination that is usually not compatible with most biological samples, the incorporation of radionuclide-tagged prosthetic groups into biomolecules becomes the method of choice.
However, the widespread use of 18F-labeled biomolecules such as peptides and proteins for positron emission tomography (PET) is hampered by the limited availability of suitable labeling tags like N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB), also identified as in the Figures as [18F]4. Because [18F]SFB can react with primary amines of biomolecules, it has been demonstrated to be a suitable and versatile 18F-prosthetic group to radiolabel peptides, proteins, and antibodies in terms of in vivo stability and radiolabeling yield. However, the tedious synthesis procedures to obtain this compound set forth in the prior art hamper its widespread use for radiolabeling biomolecules as PET probes. Several prior available representative procedures of preparing [18F]SFB are summarized in Table 1 (FIG. 1).
Most of the radiochemical syntheses of [18F]SFB described in the literature require two to three reactors and multiple SPE or HPLC purifications. Referring to FIG. 2, Two previously described precursors, (4-ethoxycarbonyl-phenyl)trimethylammonium triflate (1a) (Wester H J, Schottelius M. Fluorine-18 Labeling of Peptides and Proteins. PET Chemistry—The Driving Force in Molecular Imaging, Schubiger P A (ed), Lehmann L (ed), Friebe M (ed). Springer: Berlin Heidelberg, (2007); Haka M S, Kilbourn M R, Watkins G L, Toorongian S A. J Label Compd Radiopharm; 7: p 823-833 (1989); Guhlke S, Coenen H H, Stöcklin G. Appl Radiat Isot; 45, p 715-727 (1994) and (4-tert-butoxycarbonyl-phenyl)trimethylammonium triflate (1b) (Wester H J, Hamacher K, Stöcklin G. Nucl Med Biol, 23, p 365-372 (1996); Hostetler E D, Edwards W B, Anderson C J, Welch M J J Label Compd Radiopharm, 42, p S720-S722 (1999), were compared in terms of radiochemical yield (RCY) and chemical as well as radiochemical purity. This comparison was repeated after the deprotection step in both pathways. It was found that the use of 1a was superior to 1b.
Although module-assisted [18F]SFB production in various semi-automated synthesizers has been reported, there are still several drawbacks for its general use. In particular, the need for two solid phase extraction (SPE) steps with three different cartridges as published by Wester et al. 6. (Wester H J, Schottelius M. Fluorine-18 Labeling of Peptides and Proteins. PET Chemistry—The Driving Force in Molecular Imaging, Schubiger P A (ed), Lehmann L (ed), Friebe M (ed). Springer: Berlin Heidelberg, (2007); Wester H J, Hamacher K, Stöcklin G. Nucl Med Biol, 23, p 365-372 (1996)) is very demanding. Various implementations include modifications of commercial synthesizers to perform the SPE steps. These hardware and software changes make it difficult for labs lacking engineering expertise and facilities to repeat the procedures. Recently, Kabalka et al. (Kabalka G, J Label Compd Radiopharm, 51, p 68-71 (2008)) described an efficient preparation of [18F]SFB based on a three-step, one-pot procedure. The entire process takes about 60 minutes and the deprotection/hydrolysis step is carried out with an aqueous tetrapropylammonium hydroxide solution. Subsequent time-consuming azeotropic drying is necessary due to the application of aqueous reagent. Another approach published by Carroll et al. (Carroll M, J Nucl Med, 49(S): p 298P (2008)) is to utilize iodonium salt precursor for preparing [18F]SFB in a one-step, one-pot procedure. Although it is a very attractive route, the overall yield is low and the precursor is also unstable. Therefore, further improvement and simplifying [18F]SFB synthesis would be very beneficial for use in an automated process.